Aminated isoflavonoid derivatives and uses thereof

ABSTRACT

Aminated isoflavonoid synthesized by aminating the 4-keto group of isoflavanone and isoflavanone ring systems, pharmaceutical compositions containing same and uses thereof as therapeutic agents.

This application is a 371 of PCT/AU2003/001446, filed Nov. 3, 2003; thedisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to aminated isoflavonoidderivatives based on nitrogen substitution of the 4-keto group ofisoflavone and isoflavanone ring systems. The present invention furtherrelates to the synthesis of the aminated isoflavonoid derivatives,compositions containing same and uses thereof as therapeutic agents.

BACKGROUND OF THE INVENTION

Naturally-occurring plant isoflavones are known to possess a wide rangeof fundamental biological effects on human cells includinganti-oxidation and the up-regulation and down-regulation of a widevariety of enzymes and signal transduction mechanisms. Mitotic arrestand cytotoxicity of human cancer cells, increased capillarypermeability, increased cellular adhesion, increased response ofvascular smooth muscle cells to vaso-relaxants, and agonism of estrogenreceptors, are just a few examples of the responses of animal cells tothe biological effects of naturally-occurring isoflavonoids.

A range of therapeutic benefits as a result of these biological outcomeshave been identified including the treatment and prevention ofpre-menopausal symptoms such as pre-menstrual syndrome, endometriosis,uterine fibroids, hyperlipidaemia, cardiovascular disease, menopausalsymptoms such as osteoporosis and senile dementia, alcoholism, benignprostatic hypertrophy, and cancers such as prostate, breast and largebowel carcinomas [see WO 93/23069; WO 96/10341; U.S. Pat. No. 5,424,331;JP 62-106017; JP 62-106016; U.S. Pat. No. 5,516,528; JP 62-106016A2; JP62-106017A2; JP 61-246124; WO 98/50026; WO 99/43335; WO 00/49009; WO00/644438; WO 99/48496].

While over 700 different naturally occurring isoflavones are described,only a few are confirmed as having potential therapeutic benefits inanimals including humans. These include daidzein, genistein,formononetin, biochanin and glycitein. These and all naturally occurringisoflavones are found in nature as the monomeric form either in a freestate, or, more likely, bound to a carbohydrate moiety (glycoside). Theisoflavone has to be separated from this moiety before it becomesbiologically active.

A number of compounds with a structure related to naturally occurringplant isoflavones are also described as having biological propertieswith potential therapeutic benefit to animals including humans. Theseinclude compounds that are naturally occurring metabolites of plantisoflavones produced by bacterial fermentation by gut flora and embracecompounds such as equol and 0-desmethylangolensin [WO 93/23069; WO98/08503; WO 01/17986; WO 00/66576]. Also included in this group is thesynthetic isoflavonoid ipriflavone, which is developed for the treatmentof postmenopausal osteoporosis [WO 91/14429] and a wide range ofsynthetic isoflavonoid analogues [WO 98/08503].

Despite the considerable research and accumulated knowledge in relationto isoflavonoid compounds and derivatives thereof, the full ambit oftherapeutically useful isoflavonoid compounds and their activities isyet to be realised. Moreover, there is a continual need for new,improved or at least alternative active agents for the treatment,prophylaxis, amelioration, defence against and/or prevention of variousdiseases and disorders.

A requirement accordingly exists for new generation compounds thatexhibit physiological properties important to the health and well-beingof animals, particularly humans, and to find new methods which exploitthese properties for the treatment, amelioration and prophylaxis ofdisease.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have discovered a new class ofmolecules based on aminated isoflav-4-one and isoflavan-4-one compounds.In particular the aminated isoflavonoid compounds of the inventionrelate to imine, hydrazone, semicarbazone, azine, oxime and aminederivatives of isoflav-4-ones and isoflavan-4-ones. The activities ofthe compounds of the invention are surprising and quite unexpected, evenin light of what is presently known about the non-aminated isoflav-4-oneand isoflavan-4-one compounds from which these can be derived.

Thus according to an aspect of the present invention there is provided acompound of the general formula (I):

wherein

-   R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are independently hydrogen,    hydroxy, OR₉, OC(O)H, OC(O)R₉, OS(O)R₉, OSi(R₁₀)₃, C(O)R₁₁, CO₂R₁₂,    alkyl, haloalkyl, aryl, arylalkyl, thio, alkylthio, amino,    alkylamino, dialkylamino, nitro or halo, or any two of the    substituents R₂ R₃ and R₄ together with the carbon atoms to which    they are attached form a cyclic alkyl, cyclic heteroalkyl, aryl or    heteroaryl structure,-   R₉ is alkyl, haloalkyl, aryl, arylalkyl or alkylaryl,-   R₁₀ is independently hydrogen, alkyl or aryl,-   R₁₁ is hydrogen, alkyl, aryl, arylalkyl, arylalkyl or an amino acid,    and-   R₁₂ is hydrogen, alkyl, haloalkyl, aryl, arylalkyl or alkylaryl,-   X is O, NR₁₂ or S,-   Z is R₁₃, NR₁₄R₁₅, NR₁₃CONR₁₄R₁₅, N═CR₁₆R₁₇ or OR₁₃,-   R₁₃, R₁₄ and R₁₅ are independently hydrogen, amino, thio, nitro,    cyano, or optionally substituted alkyl, haloalkyl, acyl, aryl,    heteroaryl, arylalkyl or alkylaryl, or the substituents R₁₄ and R₁₅    together with the nitrogen atom to which they are attached form an    optionally substituted cyclic heteroalkyl or heteroaromatic    structure, and-   R₁₆ and R₁₇ are independently hydrogen, amino, thio, nitro, cyano,    or optionally substituted alkyl, haloalkyl, acyl, aryl, heteroaryl,    arylalkyl or alkylaryl, or the substituents R₁₆ and R₁₇ taken    together with the carbon atom to which they are attached form an    optionally substituted isoflavonoid ring system,    or when X is NR₁₂, the substituent R₁₂ may be a bond such that R₈    and X together with the carbon atoms to which they are attached form    one of the following structures:

where Y is

and whereinR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and Z are as defined above, andthe drawing

represents either a single bond or a double bond,which compounds include pharmaceutically acceptable salts andderivatives thereof.

According to another aspect of the present invention there is provided aprocess for the preparation of a compound of formula (I) comprising thestep of reacting the 4-keto group of a compound of the formula (X):

whereinR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and X are as defined above, andthe drawing

represents either a single bond or a double bond,with an aminating agent.

It has surprisingly been found by the inventors that the aminatedisoflavonoid derivatives of the general formula (I) have particularutility and effectiveness in the regulation of a range of moleculartargets in animal cells, and that these molecular targets are intimatelyinvolved in signal transduction processes that are fundamental tocritical cellular processes such as cell growth, differentiation,migration, and death.

The aminated compounds of the present invention are found to regulate awide variety of signal transduction processes within animal cells andthat these signal transduction processes are involved in a wide range offunctions that are vital to the survival and function of all animalcells. Therefore, these compounds have broad-ranging and importanthealth benefits in animals including humans, and in particular have thepotential to prevent and treat important and common human diseases,disorders and functions.

The particular benefits of this invention lie in (a) the large range ofsignal transduction processes targeted by the compounds, (b) the factthat regulation of these various processes includes both up-regulationof some processes and down-regulation of others, and (c) that such abroad and varied effect on signal transduction processes also isaccompanied by an independent effect on a range of important enzymesthat are fundamental to metabolism and steroidogenesis.

Thus, according to another aspect of the present invention there isprovided a method for the treatment, prophylaxis or amelioration of adisease or disorder which method includes the step of administering atherapeutically effective amount of one or more compounds of formula (I)or a pharmaceutically acceptable salt or derivative thereof to asubject.

In particular the present invention provides a method for the treatment,prevention or amelioration of diseases associated with aberrant cellsurvival, aberrant cell proliferation, abnormal cellular migration,abnormal angiogenesis, abnormal estrogen/androgen balance, dysfunctionalor abnormal steroid genesis, degeneration including degenerative changeswithin blood vessel walls, inflammation, and immunological imbalance,which comprises administering to a subject one or more compounds of theformula (I) or a pharmaceutically acceptable salt or derivative thereofoptionally in association with a carrier and/or excipient.

In accordance with another aspect of the present invention there isprovided a method of inducing apoptosis in cells expressing abnormalprosurvival phenotype which comprises contacting said cells with one ormore compounds of the formula (I) or a pharmaceutically acceptable saltor derivative thereof optionally in association with a carrier orexcipient.

In accordance with another aspect of the present invention there isprovided a method for inhibiting migration of cells having an abnormalcellular migration phenotype which comprises contacting said cells witha compound of the formula (I) or a pharmaceutically acceptable salt orderivative thereof optionally in association with a carrier orexcipient.

In accordance with another aspect of the present invention there isprovided a method for inhibiting angiogenesis in tissue expressingaberrant angiogenic phenotype which comprises contacting said tissuewith a compound of the formula (I) or a pharmaceutically acceptable saltor derivative thereof optionally in association with a carrier orexcipient.

In accordance with another aspect of the present invention there isprovided a method for the treatment, prevention or amelioration ofcancer in a mammal which method comprises the step of bringing acompound of formula (I) or a pharmaceutically acceptable salt orderivative thereof into contact with cancerous tissue in a mammal thatis suffering from a tumour, such that neoplastic development in saidcancerous tissue is retarded or arrested.

According to another aspect of the present invention there is providedthe use of one or more compounds of formula (I) or a pharmaceuticallyacceptable salt or derivative thereof in the manufacture of a medicamentfor the treatment of a disease or disorder.

According to another aspect of the present invention there is providedan agent for the treatment, prophylaxis or amelioration of a disease ordisorder which agent comprises one or more compounds of formula (I) or apharmaceutically acceptable salt or derivative thereof.

According to another aspect of the present invention there is provided apharmaceutical composition which comprises one or more compounds offormula (I) or a pharmaceutically acceptable salt or derivative thereofin association with one or more pharmaceutical carriers, excipients,auxiliaries and/or diluents.

According to another aspect of the present invention there is provided adrink or food-stuff, which contains one or more compounds of formula (I)or a pharmaceutically acceptable salt or derivative thereof.

In a particularly preferred embodiment the aminated compounds of thepresent invention exhibit the following therapeutic activities:

-   1. Direct anticancer function via signal transduction inhibition,    cell cycle regulation and apoptosis induction.-   2. Prevention of cancer onset and proliferation via COX inhibition-   3. Prevention of cancer onset and proliferation via specific    5′alphareductase inhibition-   4. Anti-inflammatory effects.

These and other aspects of the invention will become evident from thedescription and claims which follow, together with the accompanyingdrawings.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents cytotoxicity curves for Cpd. 1 and Cpd. 3 againstselected tumor lines LNCaP, DU145 and NCI-H460 as follows:

Graph A Cpd. 1/LNCaP Graph B Cpd. 1/DU145 Graph C Cpd. 3/LNCaP Graph DCpd. 3/NCI-H460

FIG. 2 represents the inhibition of testosterone induced LNCaPproliferation by Cpd. 1 (Graph A), and Cpd. 2 and Cpd. 3 (Graph B).

FIG. 3 represents an inhibition profile of testosterone-induced LNCaPproliferation for Cpd. 1, Cpd. 2 and Cpd. 3.

FIG. 4 represents the inhibition of COS (PGE 3) and thromboxane synthase(TBXZ) activity by Cpd. 1, Cpd. 2 and Cpd. 3

FIG. 5 represents the ¹H n.m.r. spectrum (d6-acetone) of Cpd. 1.

FIG. 6 represents the ¹H n.m.r. spectrum (d3-acetonitrile) of Cpd. 2.

FIG. 7 represents the ¹H n.m.r. spectrum (d3-acetonitrile) of Cpd. 3.

DETAILED DESCRIPTION OF THE INVENTION

The aminated compounds of the present invention are based on isoflavonecompounds and derivatives thereof. The term “isoflavone” as used hereinis to be taken broadly to include ring-fused benzopyran molecules havinga pendent phenyl group from the pyran ring based on a1,2-diphenylpropane system. Thus, the classes of compounds generallyreferred to as isoflavones, isoflavenes, isoflavans, isoflavanones,isoflavanols and the like are generically referred to herein asisoflavones, isoflavone derivatives or isoflavonoid molecules, compoundsor derivatives.

The term “alkyl” is taken to include straight chain, branched chain andcyclic (in the case of 5 carbons or greater) saturated alkyl groups of 1to 10 carbon atoms, preferably from 1 to 6 carbon atoms, such as methyl,ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl,pentyl, cyclopentyl, and the like. The alkyl group is more preferablymethyl, ethyl, propyl or isopropyl. The alkyl group may optionally besubstituted by one or more of fluorine, chlorine, bromine, iodine,carboxyl, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylamino-carbonyl,di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl, C₁-C₄-alkoxy, formyloxy,C₁-C₄-alkyl-carbonyloxy, C₁-C₄-alkylthio, C₃-C₆-cycloalkyl or phenyl.

The term “alkenyl” is taken to include straight chain, branched chainand cyclic (in the case of 5 carbons or greater) hydrocarbons of 2 to 10carbon atoms, preferably 2 to 6 carbon atoms, with at lease one doublebond such as ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl,2-methyl-1-propenyl, 2-methyl-2-propenyl, and the like. The alkenylgroup is more preferably ethenyl, 1-propenyl or 2-propenyl. The alkenylgroups may optionally be substituted by one or more of fluorine,chlorine, bromine, iodine, carboxyl, C₁-C₄-alkoxycarbonyl,C₁-C₄-alkylamino-carbonyl, di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl,C₁-C₄-alkoxy, formyloxy, C₁-C₄-alkyl-carbonyloxy, C₁-C₄-alkylthio,C₃-C₆-cycloalkyl or phenyl.

The term “alkynyl” is taken to include both straight chain and branchedchain hydrocarbons of 2 to 10 carbon atoms, preferably 2 to 6 carbonatoms, with at least one triple bond such as ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, and the like. The alkynyl group ismore preferably ethynyl, 1-propynyl or 2-propynyl. The alkynyl group mayoptionally be substituted by one or more of fluorine, chlorine, bromine,iodine, carboxyl, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylamino-carbonyl,di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl, C₁-C₄-alkoxy, formyloxy,C₁-C₄-allyl-carbonyloxy, C₁-C₄-alkylthio, C₃-C₆-cycloalkyl or phenyl.

The term “aryl” is taken to include phenyl, biphenyl and naphthyl andmay be optionally substituted by one or more C₁-C₄-alkyl, hydroxy,C₁-C₄-alkoxy, carbonyl, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylcarbonyloxy,nitro or halo.

The term “heteroaryl” is taken to include five-membered and six-memberedrings which include at least one oxygen, sulfur or nitrogen in the ring,which rings may be optionally fused to other aryl or heteroaryl ringsincluding but not limited to furyl, pyridyl, pyrimidyl, thienyl,imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl,quinolyl, isopuinolyl, purinyl, morpholinyl, oxazolyl, thiazolyl,pyrrolyl, xanthinyl, purine, thymine, cytosine, uracil, and isoxazolyl.The heteroaromatic group can be optionally substituted by one or more offluorine, chlorine, bromine, iodine, carboxyl, nitro,C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylamino-carbonyl,di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl, C₁-C₄-alkoxy, formyloxy,C₁-C₄-alkyl-carbonyloxy, C₁-C₄-alkylthio, C₃-C₆-cycloalkyl or phenyl.The heteroaromatic can be partially or totally hydrogenated as desired.

The term “halo” is taken to include fluoro, chloro, bromo and iodo,preferably fluoro and chloro, more preferably fluoro. Reference to forexample “haloalkyl” will include monohalogenated, dihalogenated and upto perhalogenated alkyl groups. Preferred haloalkyl groups aretrifluoromethyl and pentafluoroethyl.

Optionally substituted groups are those groups where one or morehydrogens have independently been replaced by hydroxy, alkoxy, acyl,thio, aklyl thio, cyano, nitro, amino, alkylamino, dialkylamino, halo orcarboxy.

The present inventors have discovered a new class of molecules based onaminated isoflavonoid derivatives. The invention relates to thesubstitution of the 4-keto group of isoflavone and isoflavanonecompounds by nitrogen-based moieties. In particular the aminatedisoflavone derivatives relate to imines, hydrazones, semicarbazones,azines and oximes as depicted by the general formulae (II)-(VIII):

whereinR₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are independently hydrogen, hydroxy,OR₉, OC(O)R₉, OS(O)R₉, alkyl, aryl, arylalkyl, thio, alkylthio, bromo,chloro or fluoro,R₉ is alkyl, fluoroalkyl or arylalkyl,R₁₃, R₁₄ and R₁₅ are independently hydrogen, amino, cyano, thio, nitro,or optionally substituted alkyl, haloalkyl, acyl, aryl, arylalkyl oralkylaryl, or the substituents R₁₄ and R₁₅ together with the nitrogenatom to which they are attached form an optionally substituted cyclicheteroalkyl or heteroaromatic structure,R₁₆ and R₁₇ are independently hydrogen, amino, cyano, thio, nitro oroptionally substituted alkyl, haloalkyl, acyl, aryl, arylalkyl oralkylaryl, or the substituents R₁₆ and R₁₇ taken together with thecarbon atom to which they are attached form an optionally substitutedisoflavonoid ring system, andthe drawing

represents either a single bond or a double bond;more preferably they have the following substituents whereinR₁ is hydrogen,R₂, R₃, R₅, R₆ and R₉ are independently hydrogen, hydroxy, OR₉, OC(O)R₉,alkyl, aryl or arylalkyl,R₄ and R₇ are independently hydroxy, OR₉ or OC(O)R₉,R₉ is methyl, ethyl, propyl, isopropyl or trifluoromethyl, andR₁₃, R₁₄ and R₁₅ are independently hydrogen, methyl, ethyl, propyl,isopropyl, trifluoromethyl or optionally substituted phenyl, naphthyl orbenzyl, or the substituents R₁₄ and R₁₅ together with the nitrogen atomto which they are attached form an optionally substituted cyclicheteroalkyl or heteroaromatic structure,R₁₆ and R₁₇ are independently hydrogen, methyl, ethyl, propyl,isopropyl, trifluoromethyl or optionally substituted phenyl, naphthyl orbenzyl, or the substituents R₁₆ and R₁₇ taken together with the carbonatom to which they are attached form an optionally substitutedisoflavonoid ring system, andthe drawing

represents either a single bond or a double bond; andmost preferably they have the following substituents whereinR₁ is hydrogen,R₂, R₃, R₅, R₆ and R₈ are independently hydrogen, hydroxy, OR₉, OC(O)R₉or methyl,R₄ and R₇ are independently hydroxy, OR₉ or OC(O)R₉,R₉ is methyl,R₁₃ is hydrogen, methyl, ethyl, trifluoromethyl, phenyl, chlorophenyl,nitrophenyl, toluyl, naphthyl, benzyl, chlorobenzyl, nitrobenzyl ormethylbenzyl,R₁₄ is hydrogen and R₁₅ is hydrogen, methyl, ethyl, trifluoromethyl,phenyl, chlorophenyl, nitrophenyl, toluyl, naphthyl, benzyl,chlorobenzyl, nitrobenzyl or methylbenzyl, or the substituents R₁₄ andR₁₅ together with the nitrogen atom to which they are attached form anoptionally substituted cyclic heteroalkyl or heteroaromatic structure,R₁₆ and R₁₇ are independently hydrogen, methyl, ethyl, trifluoromethyl,phenyl, chlorophenyl, nitrophenyl, toluyl, naphthyl, benzyl,chlorobenzyl, nitrobenzyl or methylbenzyl, or the substituents R₁₆ andR₁₇ taken together with the carbon atom to which they are attached forman optionally substituted isoflavonoid ring system, andthe drawing

represents a single bond.

Most preferably the novel aminated isoflavonoid of formula (I) arecompounds (1) -(14) as follows:

The compounds of the invention include all salts, such as acid additionsalts, anionic salts and zwitterionic salts, and in particular includepharmaceutically acceptable salts.

Chemical functional group protection, deprotection, synthons and othertechniques known to those skilled in the art may be used whereappropriate to aid in the synthesis of the compounds of the presentinvention, and their starting materials.

The preferred compounds of the present invention also include allderivatives with physiologically cleavable leaving groups that can becleaved ill Vivo from the isoflavone or derivative molecule to which itis attached. The leaving groups include acyl, phosphate, sulfate,sulfonate, and preferably are mono-, di- and per-acyl oxy-substitutedcompounds, where one or more of the pendant hydroxy groups are protectedby an acyl group, preferably an acetyl group. Typically acyloxysubstituted isoflavones and derivatives thereof are readily cleavable tothe corresponding hydroxy substituted compounds. In addition, theprotection of functional groups on the isoflavone compounds andderivatives of the present invention can be carried out by wellestablished methods in the art, for example as described in T. W.Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, NewYork, 1981.

Most preferred isoflavone and isoflavanone starting compoundscontemplated for use in accordance with the invention includeformnononetin, biochanin, genistein, daidzein and equol, and functionalderivatives, equivalents or analogues thereof. Similarly importantcompounds are the isoflavone metabolites including dihydrodaidzein, cis-and trans-tetrahydrodaidzein and dehydroequol, and derivatives andprodrugs thereof.

Chemical and functional equivalents of a particular isoflavone should beunderstood as molecules exhibiting any one of more of the functionalactivities of the isoflavone and may be derived from any source such asbeing chemically synthesised or identified via screening processes suchas natural product screening.

The term “pharmaceutically acceptable salt” refers to an organic orinorganic moiety that carries a charge and that can be administered inassociation with a pharmaceutical agent, for example, as acounter-cation or counter-anion in a salt. Pharmaceutically acceptablecations are known to those of skilled in the art, and include but arenot limited to sodium, potassium, calcium, zinc and quaternary amine.Pharmaceutically acceptable anions are known to those of skill in theart, and include but are not limited to chloride, acetate, citrate,bicarbonate and carbonate.

The term “pharmaceutically acceptable derivative” or “prodrug” refers toa derivative of the active compound that upon administration to therecipient, is capable of providing directly or indirectly, the parentcompound or metabolite, or that exhibits activity itself.

As used herein, the terms “treatment”, “prophylaxis” or “prevention”,“amelioration” and the like are to be considered in their broadestcontext. In particular, the term “treatment” does not necessarily implythat an animal is treated until total recovery. Accordingly, “treatment”includes amelioration of the symptoms or severity of a particularcondition or preventing or otherwise reducing the risk of developing aparticular condition.

The amount of one or more compounds of formula (I) which is required ina therapeutic treatment according to the invention will depend upon anumber of factors, which include the specific application, the nature ofthe particular compound used, the condition being treated, the mode ofadministration and the condition of the patient. Compounds of formula(I) may be administered in a manner and amount as is conventionallypractised. See, for example, Goodman and Gilman, “The pharmacologicalbasis of therapeutics”, 7th Edition, (1985). The specific dosageutilised will depend upon the condition being treated, the state of thesubject, the route of administration and other well known factors asindicated above. In general, a daily dose per patient may be in therange of 0.1 mg to 5 g; typically from 0.5 mg to 1 g; preferably from 50mg to 200 mg. The length of dosing may range from a single dose givenonce every day or two, to twice or thrice daily doses given over thecourse of from a week to many months to many years as required,depending on the severity of the condition to be treated or alleviated.It will be further understood that for any particular subject, specificdosage regimens should be adjust over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions. Relatively shortterm treatments with the active compounds can be used to causestabilisation or shrinkage of coronary artery disease lesions thatcannot be treated either by angioplasty or surgery. Longer termtreatments can be employed to prevent the development of advancedlesions in high-risk patients.

The production of pharmaceutical compositions for the treatment of thetherapeutic indications herein described are typically prepared byadmixture of the compounds of the invention (for convenience hereafterreferred to as the “active compounds”) with one or more pharmaceuticallyor veterinary acceptable carriers and/or excipients as are well known inthe art.

The carrier must, of course, be acceptable in the sense of beingcompatible with any other ingredients in the formulation and must not bedeleterious to the subject. The carrier or excipient may be a solid or aliquid, or both, and is preferably formulated with the compound as aunit-dose, for example, a tablet, which may contain up to 100% by weightof the active compound, preferably from 0.5% to 59% by weight of theactive compound. One or more active compounds may be incorporated in theformulations of the invention, which may be prepared by any of the wellknown techniques of pharmacy consisting essentially of admixing thecomponents, optionally including one or more accessory ingredients. Thepreferred concentration of active compound in the drug composition willdepend on absorption, distribution, inactivation, and excretion rates ofthe drug as well as other factors known to those of skill in the art.

The formulations of the invention include those suitable for oral,rectal, ocular, buccal (for example, sublingual), parenteral (forexample, subcutaneous, intramuscular, intradermal, or intravenous),transdermal administration including mucosal administration via thenose, mouth, vagina or rectum, and as inhalants, although the mostsuitable route in any given case will depend on the nature and severityof the condition being treated and on the nature of the particularactive compound which is being used.

Formulation suitable for oral administration may be presented indiscrete units, such as capsules, sachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture such as to form a unit dosage. Forexample, a tablet may be prepared by compressing or moulding a powder orgranules containing the active compound, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound of the free-flowing,such as a powder or granules optionally mixed with a binder, lubricant,inert diluent, and/or surface active/dispersing agent(s). Mouldedtablets may be made by moulding, in a suitable machine, the powderedcompound moistened with an inert liquid binder.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations suitable for ocular administration include liquids, gelsand creams comprising the active compound in an ocularly acceptablecarrier or diluent.

Compositions of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theactive compounds, which preparations are preferably isotonic with theblood of the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, or intradermal injection. Suchpreparations may conveniently be prepared by admixing the compound withwater or a glycine buffer and rendering the resulting solution sterileand isotonic with the blood. Injectable formulations according to theinvention generally contain from 0.1% to 60% w/v of active compound andare administered at a rate of 0.1 ml/minute/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. Formulations suitable for vaginaladministration are preferably presented as unit dose pessaries. Thesemay be prepared by admixing the active compound with one or moreconventional solid carriers, for example, cocoa butter, and then shapingthe resulting mixture.

Formulations or compositions suitable for topical administration to theskin preferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include Vaseline,lanoline, polyethylene glycols, alcohols, and combination of two or morethereof. The active compound is generally present at a concentration offrom 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples ofsuch compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 M to 0.2 M concentration with respect tothe said active compound. See for example Brown, L., et al. (1998).

Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Panchagnula R, et al.,2000) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2M active ingredient.

Formulations suitable for inhalation may be delivered as a spraycomposition in the form of a solution, suspension or emulsion. Theinhalation spray composition may further comprise a pharmaceuticallyacceptable propellant such as carbon dioxide or nitrous oxide.

The active compounds may be provided in the form of food stuffs, such asbeing added to, admixed into, coated, combined or otherwise added to afood stuff. The term food stuff is used in its widest possible sense andincludes liquid formulations such as drinks including dairy products andother foods, such as health bars, desserts, etc. Food formulationscontaining compounds of the invention can be readily prepared accordingto standard practices.

Therapeutic methods, uses and compositions may be for administration tohumans or animals, including mammals such as companion and domesticanimals (such as dogs and cats) and livestock animals (such as cattle,sheep, pigs and goats), birds (such as chickens, turkeys, ducks), marineanimals including those in the aquaculture setting (such as fish,crustaceans and shell fish) and the like.

The active compound or pharmaceutically acceptable derivatives prodrugsor salts thereof can also be co-administered with other active materialsthat do not impair the desired action, or with materials that supplementthe desired action, such as antibiotics, antifungals,antiinflammatories, or antiviral compounds. The active agent cancomprise two or more isoflavones or derivatives thereof in combinationor synergistic mixture. The active compounds can also be administeredwith lipid lowering agents such as probucol and nicotinic acid; plateletaggregation inhibitors such as aspirin; antithrombotic agents such ascoumadin; calcium channel blockers such as verapamil, diltiazem, andnifedipine; angiotensin converting enzyme (ACE) inhibitors such ascaptopril and enalapril, and β-blockers such as propanolol, terbutalol,and labetalol. The compounds can also be administered in combinationwith nonsteriodal antiinflammatories such as ibuprofen, indomethacin,aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. Thecompounds can also be administered with corticosteroids.

The co-administration may be simultaneous or sequential. Simultaneousadministration may be effected by the compounds being in the same unitdose, or in individual and discrete unit doses administered at the sameor similar time. Sequential administration may be in any order asrequired and typically will require an ongoing physiological effect ofthe first or initial active agent to be current when the second or lateractive agent is administered, especially where a cumulative orsynergistic effect is desired.

The isoflavone and isoflavanone compounds for use in the preferredsynthetic methods of the present invention may be derived from anynumber of sources readily identifiable to a person skilled in the art.Preferably, the isoflavones are obtained in the form of concentrates orextracts from plant sources. Again, those skilled in the art willreadily be able to identify suitable plant species, however, forexample, plants of particular use in the invention include leguminousplants. More preferably, the isoflavone extract is obtained fromchickpea, lentils, beans, red clover or subterranean clover species andthe like.

Isoflavone extracts may be prepared by any number of techniques known inthe art. For example, suitable isoflavone extracts may be prepared bywater/organic solvent extraction from the plant source. It will beappreciated that an isoflavone extract may be prepared from any singletissue of a single species of plant or a combination of two or moredifferent tissues thereof. Similarly, an extract may be prepared from astarting material which contains a heterogeneous mixture of tissues fromtwo or more different species of plant.

Generally, where an isoflavone extract is prepared from plant material,the material may be comminuted or chopped into smaller pieces, partiallycomminuted or chopped into smaller pieces and contacted with water andan organic solvent, such as a water miscible organic solvent.Alternatively, the plant material is contacted with water and an organicsolvent without any pre-treatment. The ratio of water to organic solventmay be generally in the range of 1:10 to 10:1 and may, for example,comprise equal proportions of water and solvent, or from 1% to 30% (v/v)organic solvent. Any organic solvent or a mixture of such solvents maybe used. The organic solvent may preferably be a C2-10, more preferablya C1-4 organic solvent (such as methanol, chloroform, ethanol, propanol,propylene glycol, erythrite, butanol, butanediol, acetonitrile, ethyleneglycol, ethyl acetate, glycidol, glycerol dihydroxyacetone or acetone).Optionally the water/organic solvent mixture may include an enzyme whichcleaves isoflavone glycosides to the aglycone form. The mixture may bevigorously agitated so as to form an emulsion. The temperature of themix may range, for example, from an ambient temperature to boilingtemperature. Exposure time may be between one hour to several weeks. Oneconvenient extraction period is twenty-four hours at 90° C. The extractmay be separated from undissolved plant material and the organic solventremoved, such as by distillation, rotary evaporation, or other standardprocedures for solvent removal. The resultant extract containing watersoluble and non-water soluble components may be dried to give anisoflavone-containing extract, which may be formulated with one or morepharmaceutically acceptable carriers, excipients and/or auxiliariesaccording to the invention.

An extract made according to the description provided in the previousparagraphs may contain small amounts of oil which include isoflavones intheir aglycone form (referred to herein as isoflavones). This isoflavoneenriched oil, may be subject to HPLC to adjust the isoflavone ratios,or, if it is at the desired isoflavone ratio, may be dried, for examplein the presence of silica, and be formulated with one or more carriers,excipients and/or auxiliaries to give an isoflavone containing extract.Alternatively, the isoflavones contained in said small amounts of oilmay be further concentrated by addition to the oil of a non-watersoluble organic solvent such as hexane, heptane, octane acetone or amixture of one or more of such solvents. One example is 80% hexane, 20%acetone w/w having high solubility for oils but low solubility forisoflavones. The oil readily partitions into the organic solvent, and anenriched isoflavone containing extract falls out of solution. Therecovered extract may be dried, for example in an oven at 50° C. toabout 120° C., and formulated with one or more pharmaceuticallyacceptable carriers, excipients and/or auxiliaries.

It will be appreciated that the present invention also contemplates theproduction of suitable starting isoflavones, functional derivatives,equivalents or analogues thereof, by established synthetic techniqueswell known in the art. See, for example, Chang et al. (1994) whichdiscloses methods appropriate for the synthesis of various isoflavonesas starting materials.

Other suitable methods may be found in, for example, publishedInternational Patent Applications WO 98/08503 and WO 00/49009, andreferences cited therein, which are incorporated herein in theirentirety by reference.

Cellular Function

All cellular functions are under the control of a myriad of signalsderiving from either distant cells (endocrine signals), neighbouringcells (paracrine signals) or from within the same cell (autocrinesignals). These different signals work largely by stimulating the cell'sgenome (DNA) from where the appropriate cellular response is initiated.The process by which the signal is transmitted to the genome is known assignal transduction. By this we mean pathways, mostly involvingdifferent proteins, where activation of one protein catalyses theresponse of another protein, resulting finally in transcription of aparticular gene or set of genes. Homeostasis, by which we mean theintegrated functioning of cells, tissues and organs resulting in goodhealth, is the end product of hundreds, possibly thousands, of differentsignals entering the body's cells on a continuous basis.

From this signalling milieu, it is possible to divide signalsarbitrarily into those that are related to a ‘specialized function’, andthose that are related to the fundamental ability of the cell to existand to function. Examples of ‘specialized functions’ are pain perceptionby a nerve cell, production of antibodies by an immune cell,detoxification reactions by a liver cell, or formation of urine by akidney cell. Examples of ‘fundamental functions’ are cell survival orcell death, cell proliferation, cell migration, and angiogenesis. It canbe seen that the key to regulating whether or not a cell is able toperform ‘specialized functions’ is regulation of the cell's ‘fundamentalfunctions’.

The applicants have found that compounds of the formula (I) regulatemany of the ‘fundamental functions’ of the cell. The following are someexamples of the ‘fundamental functions’ that the inventors have found tobe regulated by the aminated compounds of the present invention.

1. Cell Survival/Death

In order to continue to function, including the ability to respond tospecialized functions, cells need to be continuously activatingpro-survival signal transduction mechanisms. Pro-survival mechanisms actat two main levels—those that actively promote survival and those thatactively suppress cell death (apoptosis).

Pro-survival mechanisms involve a number of different signaltransduction processes that ultimately cause transcription of certaingenes whose end-products promote cell survival. These differentprocesses involve, but are limited to, such molecular targets as MEK,ERK, and NFκB. Phenoxodiol has been found to operate across a range ofthese processes. One in particular by way of example is the enzyme,sphingosine kinase. Sphingosine kinase phosphorylates the substrate,sphingosine, to sphingosine-1-phosphate. Sphingosine-1-phosphate is animportant stimulator of pro-survival mechanisms and is over-expressed ina range of disease states characterized by increased longevity of cells.The aminated isoflavonoid derivatives down-regulate sphingosine kinaseactivity.

Apoptosis can be achieved by a number of mechanisms as follows.

-   -   (a) One such mechanism involves receptors known as ‘death        receptors’. These include receptors such as Fas/Mort, TGF and        TNRF. Activation of receptors normally is suppressed through the        production of blocking proteins such as C-flip. The aminated        isoflavonoid derivatives have been found to block the production        of C-flip, in so doing, promoting the death of cells.    -   (b) Another mechanism involves the activation of proteolytic        enzymes known as caspases. Once activated, these enzymes        autolyse the cell. The aminated isoflavonoid derivatives have        been found to up-regulate the activity of caspases.    -   (c) Another mechanism involves disruption of mitochondria        leading to the production of various pro-death factors. The        aminated isoflavonoid derivatives have been found to promote        such disruption through a direct and novel effect on the        mitochondria.

It can be seen from the above description, that the aminatedisoflavonoid derivatives are able to induce cell death in acomprehensive manner via a number of different pathways. The ability ofa single compound to have such broad and complementary effects is novel.But of considerable surprise is the finding that the aminatedisoflavonoid derivatives exert such pro-death effects in abnormal cellsonly.

That is, in normal healthy cells, the aminated isoflavonoid derivativeshave no discernible effect on these regulatory processes. Cells thatdisplay abnormal activity of these regulatory processes include but arenot limited to cells involved in such disease states as cancer,cardiovascular disease, autoimmune diseases, and diseases withimmunological, inflammatory or hyperproliferative components.

2. Cell Proliferation

The ability to divide in response to growth signals is anotherfundamental function required by normal, healthy cells.Sphingosine-1-phosphate appears to play a key role in facilitating theability of cells to divide. The act of cell division involves a numberof different enzymes as follows:

-   -   (a) the activation of topoisomerases (I and II) whose task it is        to organize DNA prior to mitosis;    -   (b) the activation of cyclin dependent kinases (CDKs) whose task        is it to move the genome through the different stages of        mitosis;    -   (c) inactivation of cyclin dependent kinase inhibitors (CDKIs)        whose task it is to inhibit mitosis through suppression of CDKs.

The aminated isoflavonoid derivatives surprisingly inhibit the 3 aboveenzyme systems, viz. topoisomerase II, CKDs and CDKIs in cells that arebehaving abnormally, particularly cells expressing abnormal prosurvivingphenotype or aberrant cell proliferation.

3. Cell Migration

It is well understood that the ability of a cell to migrate and tointeract with its neighbouring cells is fundamental to health anddisease. Sphingosine kinase and matrix-metalloproteases are keyregulators of this important cell function. The aminated isoflavonoidderivatives uniquely down-regulate both of these enzyme systems, thusdiminishing the ability of cells in a diseased state to migrate.

4. Angiogenesis

The ability to form new blood vessels is well known to be a key eventunderlying many disease states associated with hyperplasia. Sphingosinekinase is a key facilitator of this event. The aminated isoflavonoidderivatives by down-regulating this enzyme, selectively impairangiogenesis when it occurs in association with disease, and not inhealthy tissues.

These broad-ranging effects of the aminated isoflavonoid derivatives onsignal transduction mechanisms are complemented surprisingly byinhibitory effects on a wide range of enzymes, such enzymes not normallybeing regarded as part of signal transduction processes, but of thephysiology of the body in more general terms. These effects also includethe following:

5. Steroidogenesis

The aminated isoflavonoid derivatives inhibit a number of enzymesinvolved in steroidogenesis. These include but are not limited tosteroid dehydrogenase, 5-α-reductase and aromatase. People skilled inthe art would recognize that such effects would have significant impacton the production of steroid hormones including androgens, estrogens andcorticosteroids. Such effects would be regarded as someone skilled inthe art in having impact on the normal function of the male and femalereproductive tissues including the breast, ovary, uterus, endometrium,cervix, vagina, prostate and penis.

In summary, the inventors have surprisingly found that the aminatedisoflavonoid derivatives regulate a unique collection of enzymesinvolved in both general metabolism and physiological function, and insignal transduction pathways that play pivotal roles in cell survival,cell growth, cell differentiation, and cell response to inflammation andimmune modulators. Through regulation of this group of enzymes thecompounds of the invention have the capacity to (a) to prevent or totreat many forms of disease irrespective of the cause or pathogenesis ofthat disease, and (b) influence the full range of biological activitiesof the body's tissues and the way in which disease, age, environmentalinfluences and other drugs influence those activities.

Moreover, it is highly surprising and novel to find that these compoundscan cause a human breast cancer cell to undergo apoptosis and die, alsocan have such diverse effects as antagonising hypertension, redressingthe immunological and inflammatory imbalance underlying inflammatorybowel disease, reversing Type 1 diabetes, and reversing male patternbaldness.

It can readily be seen that the aminated isoflavonoid derivatives of thepresent invention would have particular relevance in the prevention andtreatment of various disease states and disorders as follows.

A. Diseases and Disorders Associated with Abnormal Response to GrowthSignals, Abnormal Cellular Proliferation, Dysfunctional Apoptosis, andAbnormal Migration Patterns (Metastasis)

These include:

-   -   1. all forms of cancer (pre-malignant, benign and malignant) in        all tissues of the body. In this regard, the compounds may be        used as the sole form of anti-cancer therapy or in combination        with other forms of anti-cancer therapy including but not        limited to radiotherapy and chemotherapy;    -   2. papulonodular skin lesions including but not limited to        sarcoidosis, angiosarcoma, Kaposi's sarcoma, Fabry's Disease    -   3. papulosquamous skin lesions including but not limited to        psoriasis, Bowen's Disease, and Reiter's Disease;    -   4. proliferative disorders of bone marrow including but not        limited to megaloblastic disease, myelodysplastic syndromes,        polycythemia vera, thrombocytosis and myelofibrosis;    -   5. hyperplastic diseases of the reproductive tract including but        not limited to benign prostatic hyperplasia, endometriosis,        uterine fibroids, and polycystic ovarian disease.        B. Diseases and Disorders Associated with Abnormal Angiogenesis

These include:

-   -   1. diseases and disorders associated with abnormal angiogenesis        affecting any tissue within the body including but not limited        to metastatic cancers, psoriasis, hemangiomas and        telangiectasia.        C. Diseases and Disorders Associated with Abnormal        Inflammatory/Immunological Responses

These include:

-   -   1. diseases and disorders associated with inflammatory reactions        of an abnormal or prolonged nature in any of the body's tissues        including but not limited to rheumatoid arthritis, tendonitis,        inflammatory bowel disease, ulcerative colitis, Crohn's Disease,        sclerosing cholangitis;    -   2. diseases and disorders associated with degenerative changes        within the walls of blood vessels including but not limited to        the syndrome known commonly as cardiovascular disease (embracing        the diseases atherosclerosis, atheroma, coronary artery disease,        stroke, myocardial infarction, post-angioplasty restenosis,        hypertensive vascular disease, malignant hypertension,        thromboangiitis obliterans, fibromuscular dysplasia);    -   3. diseases and disorders associated with abnormal immunological        responses including but limited to dermatomyositis and        scleroderma.    -   4. immunological imbalance including immune deficiency        associated with H.I.V. or other viral infective agents or        bacterial infective agents, and immune deficiency related to        immaturity or aging.        D. Diseases and Disorders Associated with Decreased Cellular        Function Including Depressed Response to Growth Signals and        Increased Rates of Cell Death

These include:

-   -   1. actinic damage characterized by degenerative changes in the        skin including but not limited to solar keratosis,        photosensitivity diseases, and wrinkling;    -   2. autoimmune disease characterized by abnormal immunological        responses including but not limited to multiple sclerosis, Type        1 diabetes, systemic lupus erythematosis, and biliary cirrhosis;    -   3. neurodegenerative diseases and disorders characterized by        degenerative changes in the structure of the neurological system        including but not limited to Parkinson's Disease, Alzheimer's        Disease, muscular dystrophy, Lou-Gehrig Disease, motorneurone        disease;    -   4. diseases and disorders associated with degenerative changes        within the eye including but not limited to cataracts, macular        degeneration, retinal atrophy.        E. Diseases and Disorders Associated with Dysfunctional or        Abnormal Steroidogenesis and Function of Reproductive Hormones

These include:

-   -   1. conditions in women associated with abnormal        estrogen/androgen balance including but not limited to cyclical        mastalgia, acne, dysmenorrhoea, uterine fibroids, endometriosis,        ovarian cysts, premenstrual syndrome, acute menopause symptoms,        osteoporosis, senile dementia, infertility;    -   2. conditions in men associated with abnormal estrogen/androgen        balance including but not limited to benign prostatic        hypertrophy, infertility, gynecomastia, alopecia hereditaria and        various other forms of baldness.

The physiological effects ascribed to the aminated isoflavonoidderivatives of the invention particularly relate to the general areas ofsignal transduction pathways, anti-cancer applications,anti-inflammatory activity and as cardio-protective agents. Moreparticularly the aminated isoflavonoid derivatives of the invention showbroad therapeutic indications including, and in particular, anti-canceractivity via signal transduction inhibition, cell cycle regulation andapoptosis induction, antiangiogenesis (MMP inhibition), signaltransduction perturbation (receptor protein tyrosine kinase inhibitor),COX inhibition, 5′-alpha reductase inhibition, cardio protectiveproperties and anti-inflammatory effects.

Specific areas of utility of the compounds of the present invention aredescribed and exemplified as follows:

Anti-Cancer:

In many western countries prostatic adenocarcinoma, secondary to lungcancer, is the most commonly diagnosed malignancy in men and the mostcommon cause of death (Landis et al., 1999; Hsing et al., 2000).Established treatment options for localized prostate cancer, includingsurgery (radical prostatectomy) and radiation therapy, are curative inonly 52-78% of cases with the remaining proportion of cases sufferingrelapse due to residual disease (Morris and Scher, 2000; Papatsoris andPapavassiliou, 2001). While androgens have an important role incontrolling the growth of the normal prostate gland, they also promoteonset of benign prostatic hyperplasia (BPH) and prostate cancerprogression by transactivating cellular proliferation genes facilitatevia the ligand bound androgen receptor (AR) (Amanatullah et al., 2000).Hence in early disease the mainstay of primary treatment options isandrogen ablation therapy utilising both surgical and/orpharmaco-therapeutic methods (Papatsoris and Papavassiliou, 2001).

Interestingly epidemiological studies on the prevalence of prostatecancer in eunuchs, who have a deficiency in 5′alphareductase, show thatthis subset of the population have a very low incidence of the disease.In the androgen-signaling cascade 5′AR is responsible for the conversionof testosterone to dihydrotestosterone which, in comparison withtestosterone, has a much stronger binding affinity for the AR and isable to elicit a stronger proliferative response (Papatsoris andPapavassiliou, 2001). As such considerable research effort has focusedon defining novel 5′AR inhibitors. There are two isoforms ofNADPH-dependent 5′alphareductase, termed types I and II, with type Iexpressed primarily in human scalp, skin and liver, and type IIexpressed primarily in the prostate. Finasteride, a type II specificinhibitor, is the only available SAR inhibitor to treat BPH, and earlyphase prostate cancer when used in combination with an anti-androgen(such as megestrol acetate). However, given the steroidal structure offinasteride and potential adverse effects, considerable research hasfocused on elucidating other nonsteroidal inhibitors of5′alpha-reductase that are clinically acceptable (Chen, et al., 2001).

Recent evidence infers that cancer initiation and progression may befacilitated via the excess production of prostaglandens in inflamedtissue (Vainio, 2001). Cyclooxygenases (COX) catalyse the conversion ofarachidonic acid (AA) to prostaglandins and thromboxanes. Supportingepidemiological studies in conjunction with laboratory studies providestrong evidence to suggest that traditional nonsteroidalanti-inflammatory drugs (NSAIDs including aspirin) and COX-2 inhibitors(celecoxib) may reduce the risk of colon cancer (Koki et al., 2002).Indeed clinical biopsies from many different malignancies consistentlyshow a significant over-expression of COX-2. The presumed anti-cancermechanism of action elicited by NSAIDs is thought to be due to theirability to inhibit the production of prostaglandins via COX-2, which candrive angiogenesis and prevent apoptosis of cancer cells (Vaino, 2001;Fosslien, 2001.)

Anti-Inflammatory:

Prostaglandins such as PGE₂ and PGI₂ and thromboxanes (TXs) such as TXA₂are fatty acid derivatives known as eicosanoids (Penglis et al. 2000).They are involved in both normal physiology and inflammatory responses.AA released from membrane phospholipids, is the primary substrate forCOX enzymes thus giving rise to eicosanoids. Regardless of the COXisotype (COX 1 and COX 2) prostaglandin (PGH₂) is the main intermediateof this reaction and it is the common precursor for downstreamprostanoid production (PGE₂, PGI₂ and TXA₂)). The potential of a testagent to demonstrate anti-inflammatory activity can be assessed bymeasuring the compound's ability to inhibit PG and TX synthesis inscreening assays. The preliminary data presented in the Examples whichfollow on the aminated isoflavones of the invention and their ability toinhibit both thromboxane synthase and COX support that this class ofmolecule has therapeutic application as an NSAID.

The invention is further illustrated by the following non-limitingExamples and accompanying drawings.

Example 1 General Synthetic Methods

1. Imine Synthesis

2. Hydrazone Synthesis

3. Semicarbizone Synthesis

4. Azine Synthesis

4.1 Azine Dimer Synthesis

5. Oxime Synthesis

6. Amine Synthesis (Reductive)

In the above general methods, the structures may be optionallysubstituted with the desired substituents, or synthons or derivativesthereof. The reactive amine compounds may be present as, for example,their hydrochloride salts and the reactions performed in the presence ofa base such as sodium acetate, or as appropriate as determined by askilled synthetic chemist.

Synthesis

Dihydrodaidzein (1 mmol) was refluxed with 3 mole equivalents of thephenylhydrazine hydrochloride and 3 mole equivalents of sodium acetate(246 mmol) in 4 ml of methanol for 6 hours.

The solution was filtered and the methanol removed under reducedpressure. The product was then purified by silica gel columnchromatography (10% ether, 90% dichloromethane), yielding between 40%and 60% of the product.

R=H

H-NMR: (d6-acetone) 8.046 (s), 8.018 (s), 7.787 (s), 7.194 (t), 7.088(d), 6.762 (m), 6.542 (d of d), 6.292 (d), 4.400 (d of d), 4.3165 (d ofd), 4.198 (s) CI-MS (+): 256

R=p-NO₂

H-NMR: (d3-acetonitrile) 8.685 (s), 8.638 (s), 8.239 (d), 8.079 (d),7.247 (t), 7.046 (m), 6.817 (d of d), 6.60 (d), 6.646 (d), 6.583 (d ofd), 4.546 (d), 4.392 (d), 4.309 (s) CI-MS (+): 298

R=p-CH₃

H-NMR: (d3-acetonitrile) 8.028 (s), 7.999 (s), 7.689 (s), 7.085 (d),7.000 (d), 6.45 (d), 6.529 (d of d), 6.285 (d), 4.388 (d of d), 4.321 (dof d), 4.180 (s), 2.220 (s) CI-MS(+): 361, 344, 256

In a similar way, the benzyl substituted compound 4, chloro phenylsubstituted compounds 6-8, pyrido compound 9 and cyano compound 10 werealso prepared.

The dimeric compound 5 was prepared by standard azine synthesis.

Reaction of dihydrodaidzein (1 mmol) with methylamine (3 mmol) affordedthe methyl imine compound 11. In a similar way the hydroxy imine 12 wasalso prepared with hydroxylamine.

Acylation of the amino derivative 13 from the reductive amination(ammonia/H₂/Raney Ni) with 3 eq. acetic anhydride and mild acid work-upgave the N-acetyl derivative 14 in near quantitative yield.

Example 2 Methods

Cell Cytotoxicity Analysis:

The method of Alley et al. (1988) was followed. Briefly, prior tocytotoxicity screening a growth curve was constructed for each tumourline to be screened to determine growth kinetics, optimal seedingdensity to yield logarithmic growth over five days, and thecorresponding lag time. Spent culture medium was aspirated from asub-confluent adherent monolayer culture (T-75), the cells trypsinisedand resuspended in a minimal volume of culture medium. After countingcells, a 96-well plate was seeded at an appropriate density (100 μl) toyield optimal growth parameters and the plate was then incubated at 37°C. under 5% CO2. After the pre-determined lag-time the plate was treatedwith either vehicle (negative control) or serial dilutions of the testcompound prepared in culture medium, and then incubated for a furtherfive days. MTT (0.5 mg/ml) prepared in PBS was added to all wells andincubated at 37° C. for ˜3 hr. Spent medium was then carefully aspiratedand DMSO (150 μt) was added to solubilise cells and the reducedformazan. Absorbance was then read on a SpectraMax plate reader at 570nm and viable cells in treated plates were expressed as a percentage ofcells in control plates.

Androgen Inhibition Studies:

The method of Negri-Cesi and Motta (1994) and Negri-Cesi et al. (1999)was followed. Briefly, LNCaP cells were cultured in RPMI supplementedwith 10% fetal calf serum and 2 mM L-glutamine at 37° C. under 5% CO₂.At day 0 a subconfluent LNCaP culture flask (80%) were harvested bytrypsinisation, washed, resuspended in RPMI media completed withcharcoal stripped fetal calf serum (RPMIi) and seeded into 12 wellplates at 30,000 cells per well (15,000 cells/mL). The plates werecultured at 37° C. with 5% CO₂ for 48 hours. On day 2, spent media wascarefully aspirated from all plates and then replenished with 2 ml RPMIicontaining either testosterone (0.5 pM) as positive control,testosterone plus finasteride (1 uM) as inhibitor control and vehicle asgrowth control (equivalent concentration of DMSO). Test plates weretreated as described above; only serial dilutions of test compoundsprepared in RPMIi were used. Concentrations of test compound were chosenbased on their cytotoxicity profile as determined in the cellcytotoxicity analysis methods section above i.e. the top concentrationused in the LNCaP proliferation assay was determined as theconcentration at which cells first appear close to 100% viable along thecytotoxicity curve. This concentration was chosen to ensure that anyinhibition of proliferation imparted by the test agent was due to theinhibition of 5′AR and not due to direct killing of the cells. It isimportant not to disturb cell monolayer during the addition oftreatments. On days 5 and 8 the process followed on day 2 was repeatedfor all plates. On day 11 spent media was aspirated from all wells,cells were washed gently with PBS (500 μl), trypsinised and the cells ineach well counted using a haemocytometer. The average cell count andstandard deviation was calculated and the result expressed as the %inhibition of testosterone-induced proliferation in comparison withvehicle control.

Thromboxane Synthase and COX Inhibition Screening Assays:

Human buffy coats were obtained from the Red Cross Blood Bank. Buffycoat (50 ml) was diluted 1:2 with sterile phosphate buffered saline(PBS), overlayed onto Lymphoprep density gradient medium and centrifugedat 800 g for 20 min. The mononuclear cell (MNC) layer was removed andwashed with PBS and monocyte enriched cells were prepared from MNC bycounter-current centrifugal elutriation. Monocytes were then resuspendedin RPMI tissue culture medium with 10% foetal calf serum at 1.5×10⁶cells/ml. Test analogues were prepared in DMSO and incubated withmonocytes at 37° for 30 min at either 10 or 100 μM. After the 30 minpre-incubation, bacterial lipopolysaccharide (LPS) was added (200 ng/ml)and cells were incubated for a further 18 h at 37° in 5% CO2. Aftercentrifugation, cell-free supernatants were then removed and assayed foreither prostaglandin or thromboxane production, as determined byradioimmunoassay. Because TXA₂ is labile in aqueous medium, TXB₂, thestable hydrolysis product of TXA₂, was measured. For each dose (0, 10,100 μM), incubations were performed in triplicate. Results are expressedas mean±SD, n=3. ANOVA followed by Newman-Keuls multiple comparisonstest was used to examine differences between doses and the controlvalues.

Cytotoxicity Analysis:

Cpd. 1 and Cpd. 3 exhibited moderate anticancer activity with Cpd. 1demonstrating activity against the prostate cancer lines LNCaP andDU-145 (FIGS. 1A and 1B; Table 1). A slightly better IC50 was observedagainst the androgen-independent prostate cancer line DU-145 (13.81 μM)when compared with LNCAP (16.25 μM), which is androgen responsive.Modest activity (>20 μM) was also observed against the other cell linestested. Like Cpd. 1, Cpd. 3 also exhibited moderate activity againstLNCaP (16.26 μM) (FIG. 1C; Table 1.), however while demonstrating someefficacy against DU-145 (19.2 μM), Cpd. 3 had activity against the largecell lung carcinoma line NCI-H460 (13.3 μM) (FIG. 1D and Table 1). Cpd.3 had the best overall cell killing activity against all cell linestested. In contrast to Cpd. 3, Cpd. 2, which has a nitro group insteadof the methyl group in position 4 of the phenyl ring, had the lowestactivity against all cell lines tested (Table 1).

Androgen Inhibition Studies:

In both studies investigating testosterone-induced proliferation ofLNCaP cells a two-four fold induction was observed in the rate of growthof these cells in response to testosterone (FIGS. 2A and B). Thistestosterone-induced proliferation was potently blocked by finasteride(1 μM) (FIGS. 2A and B). Taken together these data demonstrate that thescreening model is functioning. Of the 3 analogues tested, Cpd. 1 wasthe least potent (2.25 μM) inhibitor of testosterone-inducedproliferation, while Cpd. 3 exhibited a 3 fold better result (IC50 0.68μM) over Cpd. 1 (FIGS. 2, 3 and Table 2). Cpd. 2 was the most effectiveanalogue at inhibiting testosterone-induced proliferation, with no IC50determined at 37 nM, which is some 60-fold more effect than Cpd. 1(FIGS. 2, 3 and Table 2). It is noted that Cpd. 2 having anelectron-withdrawing group (—NO₂) in position 4 in comparison with anelectron-donating group (—CH₃) (Cpd. 3) enhances the inhibitory activityof the molecule in this particular assay.

Example 3 Results and Discussions

TABLE 1 Cytotoxicity comparison of Cpd. 1, Cpd. 2 and Cpd. 3 against PC3(AR negative prostate Ca), LNCaP (AR positive Prostate Ca), DU145 (ARnegative prostate Ca), MDA-MB-468 (ER negative breast Ca) and NCI-H460(large cell lung Ca) Cell Line (uM) PC3 LNCaP DU-145 MDA-MB-468 NCI-H460Analogue Avg. St. dev Avg. St. dev Avg. St. dev Avg. St. dev Avg. St.dev Cpd. 1 33.9 2.26 16.2 .074 13.8 2.12 34.9 19.3 27.6 0.60 Cpd. 2 44.83.39 50.9 4.66 46.0 0.74 26.1 1.20 32.7 0.30 Cpd. 3 27.9 6.43 16.2 3.8819.1 1.62 22.4 5.52 13.3 1.62

TABLE 2 Phenylhydrazone analogue inhibition profile oftestosterone-induced LNCaP proliferation Analogue IC50 (uM) Cpd. 1 2.25Cpd. 2 ND @ 0.035 Cpd. 3 0.68Inhibition of Thromboxane Synthase and COX:

Cpd. 1 and Cpd. 3 exhibited 100% inhibition of COX activity when assayedat 10 μM, while Cpd. 2 inhibited this activity by 77% (FIG. 4).Likewise, 10 μM Cpd. 1, Cpd. 2 and Cpd. 3 inhibited thromboxane synthaseby 33, 27 and 60% respectively but their inhibitory effect was lesspotent in comparison with their inhibition of COX. These data supportthe finding that the aminated isoflavone analogues are inhibitors ofthromboxane synthase and COX and as such, molecules with this scaffoldexhibit potential as anti-inflammatory agents. In addition, therapeuticapplication as NSAIDS inhibition of COX activity supports that thephenylhydrazone analogues also exhibit anti-cancer activity.

Cpd. 3 and Cpd. 1 were found to be the more effective anticancer agentswith respect to their direct killing ability, however, they were lesseffective at inhibiting testosterone-induced proliferation. The mosteffective analogue inhibiting testosterone-induced proliferation wasCpd. 2, which was the least effective analogue in the directcell-killing assay. These results show that there are variations on theactivities and modes of action in the two anti-cancer screens the testcompounds were subjected to. Further, the ability of these aminatedanalogues to inhibit COX confirms that this class of molecule hasmultiple anti-cancer applications and their ability to inhibitthromboxane synthase suggests a role in anti-inflammation.

The invention has been described herein, with reference to certainpreferred embodiments, in order to enable the reader to practice theinvention without undue experimentation. However, a person havingordinary skill in the art will readily recognise that many of thecomponents and parameters may be varied or modified to a certain extentwithout departing from the scope of the invention. Furthermore, titles,headings, or the like are provided to enhance the reader's comprehensionof this document, and should not be read as limiting the scope of thepresent invention.

The entire disclosures of all applications, patents and publications,cited herein, if any, are hereby incorporated by reference.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification individually or collectively, andany and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in the field ofendeavour.

REFERENCES

-   Alley M C, Scudiero D A, Monks A, Hursey M L, Czerwinski M J, Fine D    L, Abbott B J, Mayo J G, Shoemaker R H, Boyd M R. 1988 Feasibility    of drug screening with panels of human tumor cell lines using a    microculture tetrazolium assay. Cancer Res. 48: 589-601.-   Amanatullah D F, Reutens A T, Zafonte B T, Fu M, Mani S, Pestell    R G. 2000 Cell-cycle dysregulation and the molecular mechanisms of    prostate cancer. Front. Biosci., 5: D372-90.-   Chen G S, Chang C S, Kan W M, Chang C L, Wang K C, Chern J W 2001    Novel lead generation through hypothetical pharmacophore    three-dimensional database searching: discovery of isoflavonoids as    nonsteroidal inhibitors of rat 5 alpha-reductase. J Med Chem 44,    3759-63.-   Hsing A W, Tsao L, Devesa S S. 2000 International trends and    patterns of prostate cancer incidence and mortality. Int. J. Cancer    85, 60-7.-   Fosslien, E. 2001 Molecular pathology of COX-2 in cancer-induced    angiogenesis. Ann. Clin. Lab. Science. 31, 325-348.-   Koki, A. T. and Masferrer, J. L. 2002 Celecoxib: A specific COX-2    Inhibitor with anticancer properties. Cancer Control 9, Supp. 28-35.-   Landis S H, Murray T, Bolden S, Wingo P A. 1999 CA Cancer J. Clin.    49, 8-31.-   Morris, M. J. and Scher, H. I 2000 Novel strategies and therapeutics    for the treatment of prostate carcinoma. Cancer 89, 1329-1348.-   Negri-Cesi, P., Motta, M., 1994 Androgen metabolism in the human    prostatic cancer cell line LNCaP. 1994 J. Steroid Biochem. Molec.    Biol. 51, 89-96.-   Negri-Cesi, P., Colciago, A., Poletti, A. and Motta, M. 1999    5a-Reductase isozymes and aromatase are differentially expressed and    active in the androgen-independent human prostate cancer cell lines    DU145 and PC3. The Prostate 41, 224-232.-   Papatsoris, A. G and Papavassiliou, A. G. 2001 Prostate cancer:    horizons in the development of novel anti-cancer strategies. Curr.    Med. Chem.—Anti-cancer agents. 1, 47-70.-   Penglis, P. S., Cleland, L. G., Demasi, M., Caughey, G. E. and    James, M. J. 2000. Differential regulation of prostaglandin E2 and    thromboxane A2 production in human monocytes: implications for the    use of COX inhibitors. J. Immunol. 165, 1605-11.-   Vaino, H. 2001 Is Cox inhibition a panacea for cancer prevention.    Int. J. Cancer 94, 613-614.

1. A compound of formula (III) or (VIII):

wherein R₁ is hydrogen, R₂, R₃ and R₄ are independently hydrogen,hydroxy or OR₉, R₇ is hydroxy, R₅, R₆ and R₈ are independently hydrogen,hydroxy, or C₁₋₆ alkyl, R₉ is C₁₋₆ alkyl, one of R₁₄ and R₁₅ ishydrogen, the other one of R₁₄ and R₁₅ is hydrogen or is phenyl, benzyl,or pyridyl optionally substituted by one of C₁₋₄ alkyl, nitro, OR₉, haloor cyano, and the drawing “- - -” represents either a single bond or adouble bond, or a pharmaceutically acceptable salt thereof.
 2. Acompound according to claim 1, wherein R₂, R₃, and R₄ are independentlyhydrogen, hydroxyl, or methoxy, R₅ and R₆ are hydrogen, R₈ is hydrogenor C₁₋₆ alkyl, and the other one of R₁₄ and R₁₅ is hydrogen or isphenyl, benzyl or pyridyl optionally substituted by one of methyl,nitro, methoxy, chloro or cyano, or a pharmaceutically acceptable saltthereof.
 3. A compound according to claim 2, wherein R₂ and R₃ arehydrogen, R₄ is hydroxy or methoxy, R₈ is hydrogen or methyl, R₁₄ ishydrogen and R₁₅ is phenyl optionally substituted by one of methyl,nitro, methoxy, chloro or cyano, and the drawing “- - -” represents asingle bond, or a pharmaceutically acceptable salt thereof.
 4. Acompound according to claim 3 selected from compounds (1)-(10):4′,7-Dihydroxyisoflavanone (phenyl)hydrazone (1)4′,7-Dihydroxyisoflavanone (4-nitrophenyl)hydrazone (2)4,7-Dihydroxyisoflavanone (4-methylphenyl)hydrazone (3)4′,7-Dihydroxyisoflavanone (benzyl)hydrazone (4)4,7-Dihydroxyisoflavanone(4,7-dihydroxyisoflavanone)hydrazone (5)4′,7-Dihydroxyisoflavanone (2-chlorophenyl)hydrazone (6)4′,7-Dihydroxyisoflavanone (3-chlorophenyl)hydrazone (7)4′,7-Dihydroxyisoflavanone (4-chlorophenyl)hydrazone (8)4′,7-Dihydroxyisoflavanone (2-pyridyl)hydrazone (9)4′,7-Dihydroxyisoflavanone (4-cyanophenyl)hydrazone (10) or apharmaceutically acceptable salt thereof.
 5. A process for thepreparation of a compound of formula (III) or (VIII) as defined in claim1 comprising the step of reacting the 4-keto group of a compound of theformula (X):

wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are as defined in claim 2, Xis O, and the drawing “- - -” represents either a single bond or adouble bond, with a hydrazine aminating agent of formula H₂N—NR₁₄R₁₅,wherein one of R₁₄ and R₁₅ is hydrogen, and the other one of R₁₄ and R₁₅is hydrogen or is phenyl, benzyl, or pyridyl optionally substituted byone of C₁₋₄ alkyl, nitro, OR₉, halo or cyano.
 6. A pharmaceuticalcomposition which comprises one or more compounds of formula (III) or(VIII) as defined in claim 1 or a pharmaceutically acceptable saltthereof in association with one or more pharmaceutical carriers,excipients, auxiliaries and/or diluents.
 7. A drink or food-stuff, whichcontains one or more compounds of formula (III) or (VIII) as defined inclaim 1 or a pharmaceutically acceptable salt thereof.
 8. A compoundselected from: 4′,7-Dihydroxy-4-methylimino-isoflavan (11)4′,7-Dihydroxyisoflavanone oxime (12)4-Amino-3′,4′-dimethoxy-7-hydroxy-8-methylisoflavan (13)N-(3′,4′-dimethoxy-7-hydroxy-8-methyl-4-chromanyl)-acetamide (14) andpharmaceutically acceptable salts thereof.